Sarcocystis neurona is an apicomplexan parasite that is the primary cause of equine protozoal myeloencephalitis (EPM). Due to several factors, definitive pre-mortem diagnosis of EPM remains exceedingly difficult. In particular, the seroprevalence of S. neurona in horses is significant, yet the true incidence of EPM is quite low, thus indicating that infection does not equate with disease. Additionally, the immunoblot remains the only commercial assay available for testing samples from suspect EPM horses; while development of this test was a significant advance, it is a decade-old, first-generation assay that needs to be supplanted.
EPM is a common and debilitating infectious disease that affects the central nervous system of horses. The first detailed description of the disease was published in 1970 (Rooney et al., 1970), but it was not until 1991 that the etiological agent of EPM was isolated and designated S. neurona (Dubey et al., 1991). S. neurona is related to the human and animal pathogen Toxoplasma gondii and to the important veterinary pathogen Neospora spp. These species are phylogenetically classified into the Coccidia, which are all obligate intracellular parasites that produce a resistant oocyst during growth in the intestinal epithelium of their definitive host. Similar to other species of Sarcocystis, S. neurona has an obligatory heteroxenous life cycle, with the opossum (Didelphis virginiana) serving as a definitive host (Fenger et al., 1995). The intermediate host(s) include skunks (Cheadle et al., 2001b), raccoons (Dubey et al., 2001c), armadillos (Cheadle et al., 2001a), and cats (Dubey et al., 2000), although felids may be only an experimental intermediate host that does not contribute to the parasite life cycle in nature. Horses become infected with S. neurona by ingesting sporocysts in feces from the opossum, but unlike the normal intermediate hosts, mature sarcocysts have not been found in equine tissues (MacKay et al., 2000); consequently, the horse is currently considered an aberrant dead-end host. The geographic range of S. neurona appears to be limited to the Western Hemisphere, thus EPM primarily affects horses in the Americas.
Recent seroprevalence studies found that a significant proportion (45% to 55%) of horses have antibodies against S. neurona (Bentz et al., 1997; Blythe et al., 1997; Saville et al., 1997), suggesting that these animals are commonly exposed to the parasite. However, the incidence of EPM is estimated to be below 1% (MacKay et al., 2000), indicating that there is a clear dichotomy between simple infection with S. neurona and the occurrence of neurologic disease. In addition, early attempts at inducing disease by challenging horses with S. neurona sporocysts gave inconsistent results, and these studies were unable to authentically reproduce acute EPM (Cutler et al., 2001; Fenger et al., 1997). Consequently, it is apparent that other factors in addition to simple parasite infection are responsible for the progression to disease. It is well established that a robust cell-mediated immune response is important for controlling infections by coccidian parasites (Alexander et al., 1997; Baszler et al., 1999; Krahenbuhl and Remington, 1982), including S. neurona (Dubey et al., 2001a; Marsh et al., 1997), and it is possible that susceptibility to EPM may be increased in horses with inappropriate and/or suppressed immune responses during S. neurona infection. Accordingly, the use of stress to induce a transient immunosuppression has been shown to provide some improvement to the equine challenge model for EPM (Saville et al., 2001).
Definitive antemortem diagnosis of EPM remains exceedingly difficult, for a variety of reasons. Horses afflicted with EPM exhibit signs that are similar to a number of different neurological disorders (MacKay et al., 2000). Furthermore, S. neurona infection does not equate to disease, since only a small proportion of seropositive horses will suffer from EPM; as a consequence, the detection of anti-S. neurona antibodies in serum provides little diagnostic information other than previous exposure to the parasite. Analysis of cerebrospinal fluid (CSF) to reveal intrathecal antibody production, thus suggesting CNS infection, has improved the predictive value of antibody detection for EPM diagnosis. However, interpretation of CSF antibody presence can be confounded by contamination of the CSF sample with minute amounts of serum antibodies (Miller et al., 1999), and it is becoming apparent that the presence of antibodies in the CSF is not a definitive indication of active disease. Additionally, the contemporary diagnostic assays are hampered by several intrinsic problems, and they provide only mediocre predictive value for EPM diagnosis. Western blot analysis (a.k.a., immunoblot) of crude S. neurona lysate remains the immunodiagnostic test that is used to detect antibodies in suspect EPM horses (Granstrom et al., 1993). The continued use of the immunoblot has been necessitated by perceived antigenic cross-reactivity between different species of Sarcocystis, and the assay relies on the recognition of several antigens, primarily in the low molecular weight range, by serum/CSF antibodies (Dubey et al., 2001b; Granstrom et al., 1993; MacKay et al., 2000). Recent attempts to improve the immunoblot test have included the use of antibodies against the related parasite Sarcocystis cruzi to block cross-reactive epitopes, theoretically increasing the specificity of the immunoblot analysis for anti-S. neurona antibodies (Rossano et al., 2000). Unfortunately, western blot analysis is primarily a research tool that is relatively laborious and somewhat hindered by subjectivity, so any improvements to the immunoblot are of limited value. While the immunoblot has been utilized for a number of years to help diagnose EPM, it is a first-generation test that needs to be replaced with improved assays based on simplified, and thus more reliable, techniques that are more appropriate for diagnostic use. Nucleic acid amplification assays (polymerase chain reaction; PCR) for S. neurona detection have been developed based on the S. neurona ribosomal RNA genes (Fenger et al., 1994; Marsh et al., 1996). These PCR-based assays detect the presence of S. neurona DNA, and therefore the parasite, in the horse, so they can provide a definitive indication of active infection. However, prior to the present invention, these nucleic acid-based tests have been inherently unreliable. Specifically, parasites may be very few or non-existent in a CSF sample, so there will be no target molecules (i.e., parasite genomic DNA) for PCR amplification. More importantly, the general use of PCR for diagnosis is still suspect; although measures can be taken to improve the reliability of PCR, the technique continues to be troubled by both false positive and false negative results.
Research efforts directed toward understanding immunity against S. neurona infection and improving EPM diagnosis have been somewhat hampered by the lack of molecular information for S. neurona. The identification of S. neurona-specific antigens and characterization of the genes encoding these antigens as provided by the present invention hereby allow for the production of recombinant parasite antigens via expression in E. coli and the subsequent generation of monoclonal and monospecific polyclonal antibodies against the individual S. neurona antigens. The recombinant proteins and specific antibodies provided by the invention serve as valuable reagents for conducting immunological studies on S. neurona infections and the progression to EPM. Additionally, these reagents allow for the development of new and more reliable diagnostic tests; for example, a recombinant S. neurona antigen furnishes the key component for a simple and efficient enzyme-linked immunosorbent assay (ELISA) that can be used to monitor specific antibodies in equine serum or CSF. As provided by the teachings herein, the development of an ELISA that is based on a single recombinant S. neurona antigen rather than whole-parasite lysate provides a second-generation assay that significantly improves current methodologies for identifying S. neurona-infected animals. Notably, the use of a single antigen ELISA will allow for a more in-depth and complete dissection of antibody responses to S. neurona, which may distinguish between horses that have been simply exposed to the parasite versus horses that are actively infected and suffering from EPM.
A fluctuating equilibrium is maintained between the cell-mediated and the humoral (antibody) responses of the vertebrate immune system, and this balance will become biased, depending on the immune stimulus, in an effort to optimize the protective response. The two arms of the immune system are characterized by Th1 or Th2 lymphocytes that differ in their profile of secreted cytokines, and these immune factors target and regulate different effector cells and mechanisms. Immunoglobulin isotype switching is an important immune mechanism that allows the host to generate functional diversity in the humoral response, and the specific antibody isotype produced is largely controlled by the cytokines associated with the Th1 and Th2 balance (Finkelman et al., 1990). For example, in the mouse, a perturbation to the host that stimulates the immune system predominantly in the Th2 direction will generate an antibody response that is characterized by IgE and IgG1, whereas an immune response that is skewed towards a Th1 profile will be characterized by IgG2a and IgG3 (Finkelman et al., 1990; Snapper et al., 1997). It is generally believed that a Th1 cell-mediated response is necessary for control of coccidian parasites (Alexander et al., 1997; Krahenbuhl and Remington, 1982), so the role of antibody class switching for protection against S. neurona infection is unclear but may be secondary or unimportant. However, since the antibody isotypes produced during an infection will vary depending on the immune response that has been elicited, monitoring the relative levels of the specific isotypes will provide a means for assessing the nature of the immune response (i.e., Th1 versus Th2) in S. neurona-infected and EPM horses. The selection of an antigen for development of a diagnostic test can be somewhat subjective since any particular pathogen is composed of numerous antigenic proteins. Logically, the target molecule in a diagnostic assay must unfailingly elicit a detectable antibody response in the infected animal. A number of previous studies have demonstrated that surface antigens of the Coccidia are exceedingly immunogenic. In particular, the primary surface antigens of Toxoplasma gondii (Handman and Remington, 1980; Sharma et al., 1983) and Neaspora caninum (Howe et al., 1998) have been shown to be immunodominant. These surface antigens, designated SAGs and SAG-related sequences (SRSs), have been implicated in host cell attachment and invasion by the parasite (Dzierszinski et al., 2000; Grimwood and Smith, 1992; Hemphill, 1996; Mineo and Kasper, 1994; Mineo et al., 1993), most likely through interactions with sulfated proteoglycans on the host cell surface (He et al., 2002; Jacquet et al., 2001). In addition to their probable role as adhesins, there is increasing evidence that some of these surface antigens are involved in modulation of the host immune response (Lekutis et al., 2001). Significantly, the TgSAG1 surface antigen of T. gondii has been shown to protect mice against acute toxoplasmosis (Bulow and Boothroyd, 1991), and the NcSAG1 (p29) major surface antigen of N. caninum has been used to develop an ELISA for detection of Neospora infection in cattle (Howe et al., 2002). Collectively, these previous studies demonstrate that coccidian SAGs are at least candidate proteins for the development of both diagnostic assays and protective vaccines. Prior to the present invention, however, it had not been shown that the surface antigens of S. neurona (i.e., SnSAG2, SnSAG3, and SnSAG4) are effective target molecules for examining immune responses in infected horses and for developing improved assays for EPM diagnosis. The present invention utilizes recombinant S. neurona SAGs that are provided by the invention to provide simple and reliable ELISAs, and these assays can be used to scrutinize specific humoral immune responses in EPM horses and for detecting the presence of S. neurona in a test sample. Importantly, the developed ELISAs are valuable as tools to aid in the diagnosis of EPM infection in horses.
Nucleic acids of certain Sarcocystis and Toxoplasma species are known in the art. For example, Eschenbacher K-H et al. “Cloning and expression in Escherichia coli of cDNAs encoding a 31-kilodalton surface antigen of Sarcocystis muris”. Molec. Biochem. Parasitol. 1992, 53:159–168 (1992). Eschenbacher discloses the cloning and expression of a surface coat protein of Sarcocystis muris merozoites consisting of 280 amino acids with a predicted size of 31 kDa.
Velge-Roussel F. et al. “Intranasal Immunization with Toxoplasma gondii SAG1 induces protective cells into both NALT and GALT compartments. Infection and Immunity, 2000, 68: 969–972, discloses that intra-nasal immunization with a SAG1 protein derived from Toxoplasma gondii plus a cholera toxin provides protective immunity in mice. Specific cellular response was achieved in nasal and mesenteric compartments after i.n. immunization. T. gondii naturally invading the intestine of its host, in this case the mouse, and can be partially controlled by i.n. immunization with the protein SAG1 plus CT.
Nielsen et al. discloses the construction of a DNA vaccine using the recombinant form of the surface coat protein SAG1 in Toxoplasma gondii, consisting of 824-nucleotides encoding the 275 amino acid protein. Animals immunized with this plasmid produce anti-SAG1 antibodies which recognize the native SAG1. See, Nielsen H. V et al. “Complete protection against lethal Toxoplasma gondii infection in mice immunized with a plasmid encoding the SAG1 gene”. Infection and Immunity, 1999, 67: 6358–6363.
Peterson et al. discloses the use of an E. coli produced vaccine comprised of a recombinant Toxoplasma gondii SAG1 with alum as adjuvant, protecting mice against infection with T. gondii. Immunization with E. coli expressing rSAG1 in alum induced partial protective immunity against lethal infection with T. gondii in mice. See, Petersen E, Nielsen H V, Christiansen L, Spenter J. Immunization with E. coli produced recombinant T. gondii SAG1 with alum as adjuvant protect mice against lethal infection with Toxoplasma gondii. Vaccine. 1998 August;16(13):1283–9.
Bonenfant et al. discloses intranasal immunity with SAG1 and nontoxic mutant heat-labile enterotoxins protecting mice against Toxoplasma gondii. High level protection was assessed by the decreased load of cerebral cysts after challenge with the 76H strain of T. gondii from a group of mice immunized with LTR 72 plus SAG1 and LTK63 plus SAG1. See, Bonenfant C, Dimier-Poisson I, Velge-Roussel F, Buzoni-Gatel D, Del Giudice G, Rappuoli R, Bout D. “Intranasal immunization with SAG1 and nontoxic mutant heat-labile enterotoxins protects mice against Toxoplasma gondii”. Infect Immun. 2001 March;69(3):1605–12.
Haumont et al. discloses that a recombinant form of Toxoplasma gondii SAG1 used in vaccination had a significant protective effect against maternofetal transmission of tachyzoites. Absence of parasites in fetuses was demonstrated in 66–86% of fetuses from adult guinea pigs. There was no quantitative correlation between anti-SAG1 antibody titers and protection against maternofetal transmission. This is reference also demonstrates that a subunit vaccine based on SAG1 confers a high degree of protection against congenital T. gondii infection. Haumont M, Delhaye L, Garcia L, Jurado M, Mazzu P, Daminet V, Verlant V, Bollen A, Beaumans R, Jacquet A. “Protective immunity against congenital toxoplasmosis with recombinant SAG1 protein in a guinea pig model”. Infect Immun. 2000 September;68(9):4948–53.
Angus et al. discloses that immunization with a DNA plasmid encoding the SAG1 (p30) protein of Toxoplasma gondii is immunogenic and protective in mice. Sera of immunized mice showed recognition of T. gondii tachyzoites by immunofluorescence and exhibited high titers of antibody to SAG1 by ELISA. This data suggest that nucleic acid vaccination can provide protection against T. gondii infection in mice. See, Angus C W, Klivington-Evans D, Dubey J P, Kovacs J A.” Immunization with a DNA plasmid encoding the SAG1 (P30) protein of Toxoplasma gondii is immunogenic and protective in rodents”. J Infect Dis. 2000 January;181(1):317–24.
Fort Dodge Animal Health, “Vaccine Development” discloses that an S. neurona merozoite culture that is chemically inactivated and incorporates an adjuvant is used as an EPM vaccine. This vaccine has been conditionally licensed for use but without any indication of its effectiveness in preventing Sarcocyst neurona induced EPM Fort Dodge Animal Health, “Vaccine Development” Discloses that an S. neurona merozoite culture that is chemically inactivated and incorporates an adjuvant is used as the EPM vaccine. Fort Dodge Animal Health, 20001.
Other references of interest include:Buxton D. “Protozoan infections in sheep and goats: recent advances” Vet. Res. 1998, 29 (3–4):289–310; O,Donoghue P J et al. “Attempted immunization of swine against acute sarcocystosis using cystozooite-derived vaccines”. Vet. Immunol Immunopathol. 1985 January;8(1–2):83–92; Bulow R and Boothroyd J. C. “Protection of mice from fatal Toxoplasma gondii infection by immunization with p30 antigen in liposomes”. J. Immunol. 1991, 147 3496–3500; Dame J B, MacKay R J, Yowell C A, Cutler T J, Marsh A, Greiner E C “S. falcatula from passerine and psittacine birds: synonymy with S. neurona, agent of EPM”. J. Parasitol. 1995, December; 81(6):930–5; Mishima M, Xuan X, Shioda A, Omata Y, Fujisaki K, Nagasawa H, Mikami T. “Modified protection against Toxoplasma gondii lethal infection and brain cyst formation by vaccination with SAG2 and SRS1”. J Vet Med Sci. 2001 April;63(4):433–8; Aosai F, Mun H S, Norose K, Chen M, Hata H, Kobayashi M, Kiuchi M, Stauss H J, Yano A. “Protective immunity induced by vaccination with SAG1 gene-transfected cells against Toxoplasma gondii infection in mice”. Microbiol Immunol. 1999;43(1):87–91; Artois M, Cliquet F, Barrat J, Schumacher C L. “Effectiveness of SAG1 oral vaccine for the long-term protection of red foxes (Vulpes vulpes) against rabies”. Vet Rec.1997, Jan. 18;140(3):57–9; Follmann E H, Ritter D G, Baer G M. “Evaluation of the safety of two attenuated oral rabies vaccines, SAG1 and SAG2, in six Arctic mammals”. Vaccine. 1996 March;14(4):270–3; and Windeck T, Gross U.” Toxoplasma gondii strain-specific transcript levels of SAG1 and their association with virulence”. Parasitol Res. 1996;82(8):715–9.
Yet, despite the foregoing art, there remains a need in the art for a safe and effective vaccine against Sarcocystis neurona. Likewise, as set forth above there is also a need in the art for diagnostic kits including antigen and antibody kits for fast and reliable diagnosis of Sarcocystis neurona infection.